Telomeres, the ends of chromosomes, consist of long tandem arrays of G-rieh repeats complexed to specific proteins. Telomeres act as protective caps, shielding chromosome ends from DNA repair and DNA damage checkpoints and they serve as templates for replication by the enzyme telomerase. Telomere integrity is essential for chromosome stability and maintenance, and telomeres play a key role in replicative senescence and cancer in human cells. Telomere structure and function is regulated by the telomere repeat binding factors (TRF1 and 2) and their associated proteins. Tankyrase is a human telomeric poly(ADP-ribose) polymerase (PARP) that modifies TRF 1, inhibiting its ability to bind telomere DNA. Overexpression of tankyrase releases TRF 1 from telomeres and promotes telomere elongation in human cancer cells, indicating tankyrase as a positive regulator of telomere length. The goal of this research proposal is to elucidate the regulation and mechanism of tankyrase function at telomeres in normal and transformed human cells. First, we will determine if tankyrase is essential for telornere function by generating and characterizing stable human tumor cell and normal cell lines expressing dominant-negative or anti-sense tankyrase alleles. In addition, we will use the mouse embryonic stem cell system to eliminate one or both alleles of tankyrase. Second, we will identify tankyrase-interacting proteins and further investigate one interacting factor that is a novel substrate for tankyrase. Third, we will use an in vitro PARP assay to screen for regulators of tankyrase's PARP activity. And fmally, we will determine if tankyrase2, a closely related human homolog, is a component of the human telomeric complex. Tankyrase's PARP activity can be inhibited by small molecules, indicating it as a good target for clinical strategies to control telomere length. Since telomere length maintenance is required for the growth of tumor cells, tankyrase inhibitors may be particularly valuable for regulating cancer cell viability and may thus have important clinical relevance.